Inhibition of the MEK/ERK pathway suppresses immune overactivation and mitigates TDP-43 toxicity in a Drosophila model of ALS

TDP-43 is an important DNA/RNA-binding protein that is associated with age-related neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD); however, its pathomechanism is not fully understood. In a transgenic RNAi screen using Drosophila as a model, we uncovered that knockdown (KD) of Dsor1 (the Drosophila MAPK kinase dMEK) suppressed TDP-43 toxicity without altering TDP-43 phosphorylation or protein levels. Further investigation revealed that the Dsor1 downstream gene rl (dERK) was abnormally upregulated in TDP-43 flies, and neuronal overexpression of dERK induced profound upregulation of antimicrobial peptides (AMPs). We also detected a robust immune overactivation in TDP-43 flies, which could be suppressed by downregulation of the MEK/ERK pathway in TDP-43 fly neurons. Furthermore, neuronal KD of abnormally increased AMPs improved the motor function of TDP-43 flies. On the other hand, neuronal KD of Dnr1, a negative regulator of the Drosophila immune deficiency (IMD) pathway, activated the innate immunity and boosted AMP expression independent of the regulation by the MEK/ERK pathway, which diminished the mitigating effect of RNAi-dMEK on TDP-43 toxicity. Finally, we showed that an FDA-approved MEK inhibitor trametinib markedly suppressed immune overactivation, alleviated motor deficits and prolonged the lifespan of TDP-43 flies, but did not exhibit a lifespan-extending effect in Alzheimer disease (AD) or spinocerebellar ataxia type 3 (SCA3) fly models. Together, our findings suggest an important role of abnormal elevation of the MEK/ERK signaling and innate immunity in TDP-43 pathogenesis and propose trametinib as a potential therapeutic agent for ALS and other TDP-43-related diseases. Supplementary Information The online version contains supplementary material available at 10.1186/s12979-023-00354-8.


Drosophila strains
The following fly strains were obtained from the Bloomington Drosophila Stock Center (BDSC), UAS-lacZ (#8529), UAS-rl (#36270), GMR-Gal4 (#84247), RNAi-mCherry (#35785, a control for short hairpin RNAi knockdown), RNAi-luciferase (#31603, a control for long hairpin RNAi For neuronal expression of long hairpin RNAi lines used in this study, a copy of UAS-Dcr2 was co-expressed to boost the knockdown efficiency [1]. And, for the various RNAi flies from different sources examined in this study, the mRNA and protein levels as well as the behavioral assays are always compared to their respective backcrossed isogenized RNAi control lines as specified in each experiment. The UAS-hTDP-43 flies were described previously [2], and the TubGS and the UAS-Aβarc fly lines were the kind gifts from Dr. N. Bonini and Dr. D. Huang, respectively. For simultaneous and independent genetic manipulations, the following stable fly lines carrying multiple transgenes were generated:  Table S1. Unless otherwise noted, only male flies were tested in the age-associated behavioral assays. For adult-onset, neuronal expression of the UAS-hTDP-43 and other UAS-RNAi transgenes, the elavGS and TubGS driver were induced by supplementing the regular fly food with 80 μg/mL RU486 (TCI,. For the in vivo MEK inhibitor tests, DMSO or Trametinib (Selleck, S2673) was added in the fly food at the concentrations specified in the main text.

Fly eye degeneration assessment
To evaluate the integrity of the Drosophila eye, z-stack images of the external eyes of adult flies at indicated ages were acquired using an Olympus SZX16 stereomicroscope. The severity of the eye degeneration was assessed by rough surface, swelling and loss of pigment cells of the compound eyes. Each fly eye was single-blindly scored in a scale of 0 to 4, with 0 for no degeneration and 4 for the complete degeneration. See also Figure S1.

Climbing assay
For the climbing assay, 15-20 flies were transferred into an empty polystyrene vial and gently tapped down to the bottom of the vial. The number of flies that climbed over a distance of 3 cm within 10 seconds was recorded. The test was repeated three times for each vial and about 10 vials per group were examined.

Lifespan assay
For the lifespan experiments, 20 flies per vial and about 10 vials per group were tested. Flies were transferred to fresh fly food every 3 days and the number of dead flies in each vial was recorded. The log-rank test was used for analyzing the lifespan curves and the "50% survival" shown on the curves was derived from the compilation of all vials of the same group. The actual median lifespan of each group was calculated as the average age (day) at which 50% of the flies in a vial died. The statistical significance of the median lifespans between two or more groups was determined by Student's t-test or one-way analysis of variance (ANOVA), MEKi mitigates TDP-43 toxicity_WYue et al respectively. Flies lost prior to natural death because of escape or accidental death were excluded from the final analysis.

Protein extraction and western blotting
Fly heads or dissected fly brains were homogenized and lysed in ice cold 2% SDS lysis buffer (100 mM Tris-HCl at pH 6.8, 2% SDS, 20% glycerol, 3% DTT, 0.04% bromophenol blue) containing the protease inhibitor cocktail (Roche, 04693132001) and phosphatase inhibitor cocktail (Roche, 04906845001). Samples were sonicated, boiled at 95 °C for 5 minutes and then centrifuged at 12,000 g for 10 min at 4°C. The supernatants were then loaded in 10% SDS-PAGE (Invitrogen) and probed with the primary and secondary antibodies listed below.
The immunoblots were detected using the High-sig ECL Western Blotting Substrate (Tanon, e168230). Images were captured with an Amersham Imager 600 (GE Healthcare) and the densitometry was measured with ImageJ. The contrast and brightness were adjusted equally using Adobe Photoshop CC2019. GAPDH was used as a loading control for normalization as indicated in the figures.

Statistical analysis
Unless otherwise noted, the statistical significance in this study is determined by one-way ANOVA with Tukey's HSD post-hoc test, log-rank test (lifespan), or unpaired, two-tailed Student's t-test at *p < 0.05, **p < 0.01, and ***p < 0.001. The error bars represent the standard error of the mean (SEM). degeneration severity. Score 0: no deleterious change at all; Score 1: rough eye and less than 25% loss of pigment cells; Score 2: about 25%-50% loss of pigment cells; Score 3: about 50%-75% loss of pigment cells; Score 4: more than 75% loss of pigment cells or with obvious eye swelling and/or deformation. Intermediate scores were assigned when a sample appeared to fall between two classes. For example, score 0.5: only rough eye but no loss of pigment cells was observed. Deg., degeneration. Scale bar: 100 μm.